Diagnostic accuracy of thoracic CT to differentiate transudative from exudative pleural effusion prior to thoracentesis.

BACKGROUND: Computed tomography (CT) scan is commonly performed for pleural effusion diagnostis in the clinic. However, there are limited data assessing the accuracy of thoracic CT for the separation of transudative from exudative effusions. The study aimed to determine the diagnostic value of thoracic CT in distinguishing transudates from exudates in patients with pleural effusion. METHODS: This is a two-center retrospective analysis of patients with pleural effusion, a total of 209 patients were included from The First Affiliated Hospital of Henan University of Science and Technology as the derivation cohort (Luoyang cohort), and 195 patients from the First Affiliated Hospital of Zhengzhou University as the validation cohort (Zhengzhou cohort). Patients who underwent thoracic CT scan followed by diagnostic thoracentesis were enrolled. The optimal cut-points of CT value in pleural fluid (PF) and PF to blood CT value ratio for predicting a transudative vs. exudative pleural effusions were determined in the derivation cohort and further verified in the validation cohort. RESULTS: In the Derivation (Luoyang) cohort, patients with exudates had significantly higher CT value [13.01 (10.01-16.11) vs. 4.89 (2.31-9.83) HU] and PF to blood CT value ratio [0.37 (0.27-0.53) vs. 0.16 (0.07-0.26)] than those with transudates. With a cut-off value of 10.81 HU, the area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CT value were 0.85, 88.89%, 68.90%, 43.96%, and 95.76%, respectively. The optimum cut-value for PF to blood CT value ratio was 0.27 with AUC of 0.86, yielding a sensitivity of 61.11%, specificity of 86.36%, PPV of 78.57%, and NPV of 73.08%. These were further verified in the Validation (Zhengzhou) cohort. CONCLUSIONS: CT value and PF to blood CT value ratio showed good differential abilities in predicting transudates from exudates, which may help to avoid unnecessary thoracentesis.

Highly specific detection of Neisseria gonorrhoeae based on recombinase polymerase amplification-initiated strand displacement amplification.

Neisseria gonorrhoeae is the only pathogen that causes gonorrhea, and can have serious consequences if left untreated. A simple and accurate detection method for N. gonorrhoeae is essential for the diagnosis of gonorrhea and the appropriate prescription of antibiotics. The application of isothermal recombinase polymerase amplification (RPA) to detect this pathogen is advantageous because of its rapid performance, high sensitivity, and minimal dependency on equipment. However, this simplicity is offset by the risk of false-positive signals from primer-dimers and primer-probe dimers. In this study, RPA-initiated strand displacement amplification (SDA) was established for the detection of N. gonorrhoeae, and eliminated false-positive signals from primer-dimers and primer-probe dimers. The developed biosensor allows for the reduced generation of nonspecific RPA amplification through the design of enzyme cleavage sites on primers, introduction of SDA, and detection of the final product using a molecular beacon (MB). Using this system, the DNA double strand is transformed into single-stranded DNA following SDA, thereby providing a more suitable binding substrate and improving the efficiency of MB detection. Amplification can be conducted below 37 °C, and the process can be completed within 90 min. The limit of detection was determined to be 0.81 copies/µL. This system is highly specific for N. gonorrhoeae and exhibits no cross-reactivity with other common urogenital pathogens. The results of this study are consistent with those of real-time PCR performed on clinical specimens of urogenital secretions. In summary, the biosensor is a simple and specific detection method for N. gonorrhoeae.

Leak-proof probe for accurate detection of Neisseria gonorrhoeae by recombinase polymerase amplification-mediated lateral flow strip.

Neisseria gonorrhoeae is the only pathogen contributing to gonorrhea, a common infectious disease. Clinically, approximately 50-80% of female and 40% of male patients are asymptomatic, and these carriers are the key to gonorrhea transmission. The rapid detection of N. gonorrhoeae recessive infection is vital to curb the spread of gonorrhea. Therefore, the development of a specific, sensitive, rapid, and convenient method for the diagnosis of N. gonorrhoeae is a priority. In this study, we identified the highly conserved fitA gene of N. gonorrhoeae as a detection target through bioinformatics analysis. Then, we constructed a convenient, economical, and effective biosensor to detect N. gonorrhoeae without false-positive results based on recombinase polymerase amplification-mediated lateral flow strip by leak-proof probe. The biosensor has high sensitivity, is capable of detecting N. gonorrhoeae at concentrations as low as 102 copies/µL within 28 min, and has high specificity, which allows N. gonorrhoeae to be differentiated from other genito-urinary bacteria and fungi. Finally, this biosensor has been successfully applied to the detection of N. gonorrhoeae in clinical samples, and the results have been consistent with those determined using qRT-PCR.

Association between oral infections, triglyceride-glucose index, and in-stent restenosis.

OBJECTIVES: To investigate oral infections in patients suffering in-stent restenosis (ISR) and non-ISR and analyze the possible correlation between the oral infection and triglyceride-glucose (TyG) index, a clinical surrogate indicator of insulin resistance (IR). MATERIALS AND METHODS: A cross-sectional design was used, in which 586 patients with acute coronary syndrome who underwent coronary angiography 6-24 months after coronary stent implantation were recruited. The modified total dental index (TDI) was used to evaluate the status of oral inflammation. RESULTS: In both univariate analyses, TDI scores [3 (1.5, 4.5) vs. 2.5 (1.5, 4.0), p < 0.01] and a multivariate regression model (OR = 1.202, 95% CI = 1.085-1.333, p < 0.01), the TDI significantly correlated with ISR. The TyG index was positively associated with ISR (OR = 1.766, 95% CI = 1.055-2.957, p < 0.05). Correlation analysis showed that TDI was positively correlated with TyG index (r = 0.190, p < 0.01). Using linear regression analysis, higher TDI scores were significantly associated with IR (95% CI = 0.029-0.063, p < 0.01). CONCLUSIONS: Oral infections and TyG index were independently and positively correlated with ISR in patients with acute coronary syndrome. Oral inflammatory burden assessed by TDI score was associated with IR.

Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization.

Avian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/ß1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells. IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/ß1 integrins. The triggering of the signaling pathway-associated Integrin α5/ß1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

Cell Division Control Protein 42 Interacts With Hepatitis E Virus Capsid Protein and Participates in Hepatitis E Virus Infection.

Hepatitis E Virus (HEV) causes viral hepatitis in humans worldwide, while a subset of HEV species, avian HEV, causes hepatitis-splenomegaly syndrome in chickens. To date, there are few reports on the host proteins interacting with HEV and being involved in viral infection. Previous pull-down assay combining mass spectrometry indicated that cell division control protein 42 (CDC42), a member belonging to the Rho GTPase family, was pulled down by avian HEV capsid protein. We confirmed the direct interaction between CDC42 and avian and mammalian HEV capsid proteins. The interaction can increase the amount of active guanosine triphosphate binding CDC42 state (GTP-CDC42). Subsequently, we determined that the expression and activity of CDC42 were positively correlated with HEV infection in the host cells. Using the different inhibitors of CDC42 downstream signaling pathways, we found that CDC42-MRCK (a CDC42-binding kinase)-non-myosin IIA (NMIIA) pathway is involved in naked avian and mammalian HEV infection, CDC42-associated p21-activated kinase 1 (PAK1)-NMIIA/Cofilin pathway is involved in quasi-enveloped mammalian HEV infection and CDC42-neural Wiskott-Aldrich syndrome protein-actin-polymerizing protein Arp2/3 pathway (CDC42-(N-)WASP-Arp2/3) pathway participates in naked and quasi-enveloped mammalian HEV infection. Collectively, these results demonstrated for the first time that HEV capsid protein can directly bind to CDC42, and non- and quasi-enveloped HEV use different CDC42 downstream signaling pathways to participate in viral infection. The study provided some new insights to understand the life cycle of HEV in host cells and a new target of drug design for combating HEV infection.

Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus.

Hepatitis E, a significant global public health issue in China, is caused by sporadic infections with regional hepatitis E virus (HEV) genotypes 1, 3, and 4. To date, most immunoassays currently used to test human sera for the presence of anti-HEV antibodies cannot identify HEV at the genotype level. However, such information would be useful for identifying the source of infecting virus. Therefore, here we describe the development of a competitive enzyme-linked immunosorbent assay (ELISA) for detecting anti-genotype 1 HEV antibodies in human sera. Using recombinant genotype 1 HEV ORF3 protein as immunogen, traditional hybridoma technology was employed to generate seven monoclonal antibodies (mAbs), of which two mAbs specifically reacted with the immunogen. One of these two mAbs, 1D2, was labeled with horseradish peroxidase (HRP) for use in competitive ELISA (cELISA). After cELISA optimization using a checkerboard assay design, the amount of ORF3SAR-55 as coating antigen (100 ng/well), HRP-1D2 mAb concentration (1 µg/mL), and test serum dilution (1:10) were selected and a result ≥ 19.5 was used as the cutoff for a positive result. Importantly, cross-genotype cELISA results indicated that the cELISA could not detect anti-genotype 3 rabbit and 4 swine HEV antibodies. Moreover, human sera confirmed as negative for anti-HEV antibodies using the commercial ELISA kit were all negative via cELISA. However, because the commercial ELISA kit detects anti-all genotypes HEV antibodies and the cELISA only detects anti-genotype 1 HEV antibodies, the consistence rate of two assays detecting positive sera is low. In summary, here a cELISA for detecting anti-genotype 1 HEV antibodies was developed for use in epidemiological investigations of genotype 1 HEV infections in humans. KEY POINTS: • Seven mAbs were produced using genotype 1 HEV ORF3 protein as immunogen. • One mAb that specifically bound to genotype 1 HEV ORF3 protein was selected and labeled for use in a cELISA to detect anti-genotype 1 HEV antibodies. • The competitive ELISA developed here will aid clinical diagnosis of HEV infections and will be useful for large-scale serological testing of genotype 1 HEV infections in humans.

Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes considerable economic loss to the global pig industry. Efficient detection assay is very important for the prevention of the virus infection. Nanobodies are the advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, to develop a nanobody-based competitive ELISA (cELISA) for specifically detecting antibodies against PRRSV, three nanobodies against PRRSV-N protein were screened by camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-horseradish peroxidase (HRP) fusion protein against PRRSV-N protein was successfully constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting antibodies against PRRSV in pig sera, exhibiting a cut-off value of 23.19% and good sensitivity, specificity, and reproducibility. Importantly, the cELISA specifically detect anti-genotype 2 PRRSV antibodies. The cELISA showed more sensitive than the commercial IDEXX ELISA kit by detecting the sequential sera from the challenged pigs. The compliance rate of cELISA with the commercial IDEXX ELISA kit was 96.4%. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera. Collectively, the developed nanobody-based cELISA showed advantages of simple operation and low production cost and can be as an assay for epidemiological investigation of genotype 2 PRRSV infection in pigs and evaluation after vaccination.

Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus).

Hepatitis E virus (HEV) is a public health concern because of its zoonotic potential; however, the host species spectrum and the genetic diversity of HEV in many birds are unknown. In the present study, a novel genotype avian HEV was isolated from a bird, silkie fowl, and designated CHN-GS-aHEV (GenBank No. MN562265). The genome of CHN-GS-aHEV was analyzed in comparison with other avian HEVs' and the pathogenicity in silkie fowl was characterized. The results show that the CHN-GS-aHEV shares about 81 % identity with known avian HEV in chickens, ORF3 shares the highest identity (85.1 %-88.0 %) at the nucleotide level, while ORF2 shares the highest identity (96.5 %-98.0 %) at the amino acid level, indicating that the CHN-GS-aHEV belongs to a new genotype avian HEV. The pathogenicity study showed that silkie fowl experimentally infected with the CHN-GS-aHEV demonstrated seroconversion, viremia, fecal virus shedding, liver lesions, and increased ALT level. Furthermore, ultrastructural changes in hepatocyte cells by transmission electron microscopy were characterized by the loss of mitochondrial cristae and swollen mitochondria and endoplasmic reticulum in the infected birds, suggesting that these two organelles may play a significant role in HEV replication. Overall, this study reports the complete genome characterization of a novel avian HEV and successful experimental infection in silkie fowl, and may be serving as a prominent indicator for additional avian HEV detection in other species.

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

Fruit and vegetable consumption and its relation to risk of asthenopia among Chinese college students.

AIM: To investigate the associations between fruit and vegetable consumption and risk of asthenopia among Chinese college students. METHODS: A total of 1022 students were selected from five universities by a multi-stage stratified cluster sampling method. They were surveyed via a self-administered questionnaire including socio-demographic features, dietary and lifestyle habits, eye-related symptoms, eye care habits and history of diseases. Ascertainment of asthenopia was based on participants' subjectively reported symptoms. The associations between fruit and vegetable intake with asthenopia risk were assessed using multivariate logistic regression analysis. RESULTS: There were no significant associations between total fruit and vegetable, total vegetable, or fruit and the risk of asthenopia. Higher intake of dark-green leafy vegetable was likely to be inversely associated with asthenopia risk [odd ratio (OR): 0.60; 95%CI: 0.37-0.97; Ptrend=0.21] after controlling for nondietary and dietary risk factors. Stratified analysis showed that the inverse association between dark-green leafy vegetable intake and asthenopia risk was limited to participants with suboptimal eyesight (OR: 0.45; 95%CI: 0.25-0.82; Ptrend=0.05), wearing glasses (OR: 0.35; 95%CI: 0.17-0.72; Ptrend=0.03) or using computer ≥3h/d (OR: 0.48; 95%CI: 0.25-0.93; Ptrend=0.08). CONCLUSION: A higher consumption of dark-green leafy vegetable is associated with a lower asthenopia risk among college students with suboptimal eyesight and poor eye care habits.

Riemerella anatipestifer extracellular protease S blocks complement activation via the classical and lectin pathways.

Riemerella anatipestifer (RA) is the causative agent of infectious serositis in ducklings and other avian species. It is difficult to control the disease due to its 21 serotypes, poor cross-protection, and bacterial resistance to antimicrobial agents. The complement system is an important component of the innate immune system. However, bacterial pathogens exploit several strategies to evade detection by the complement system. Here, we purified and identified a 59-kDa RA extracellular protease S (EcpS) consisting of a gelatinase. In this study, we aimed to determine how EcpS interferes with complement activation and whether it could block complement-dependent neutrophil function. We found that EcpS potently blocked RA phagocytosis and killing by duck neutrophils. Furthermore, EcpS inhibited the opsonization of bacteria by complement 3b. EcpS specifically blocked complement 3b and complement 4b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. In summary, we show that RA can survive the bactericidal activity of the duck complement system. These results indicate that RA has evolved mechanisms to evade the duck complement system that may increase the efficiency by which this pathogen can gain access and colonize the inner tissues where it may cause severe infections.

Association among Complement Factor H Autoantibodies, Deletions of CFHR, and the Risk of Atypical Hemolytic Uremic Syndrome.

To evaluate the association among complement factor H-related (CFHRs) gene deficiency, complement factor H (CFH) autoantibodies, and atypical hemolytic uremic syndrome (aHUS) susceptibility. EMBASE, PubMed, and the ISI Web of Science databases were searched for all eligible studies on the relationship among CFHRs deficiency, anti-FH autoantibodies, and aHUS risk. Eight case-control studies with 927 cases and 1182 controls were included in this study. CFHR1 deficiency was significantly associated with an increased risk of aHUS (odds ratio (OR) = 3.61, 95% confidence interval (95% CI), 1.96, 6.63, p < 0.001), while no association was demonstrated in individuals with only CFHR1/R3 deficiency (OR = 1.32, 95% CI, 0.50, 3.50, p = 0.56). Moreover, a more significant correlation was observed in people with both FH-anti autoantibodies and CFHR1 deficiency (OR = 11.75, 95% CI, 4.53, 30.44, p < 0.001) in contrast to those with only CFHR1 deficiency. In addition, the results were essentially consistent among subgroups stratified by study quality, ethnicity, and gene detection methods. The present meta-analysis indicated that CFHR1 deletion was significantly associated with the risk of aHUS, particularly when combined with anti-FH autoantibodies, indicating that potential interactions among CFHR1 deficiency and anti-FH autoantibodies might impact the risk of aHUS.